巴西专利BR112014017474B1 system and device for use in detecting binding affinities

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专利摘要:
DEVICE FOR USE IN THE DETECTION OF CONNECTION AFFINITIES. It is a device for use in detecting binding affinities that comprises a flat waveguide (2) arranged on a substrate (3) and an optical coupler (4) for coupling coherent light (1) of a wavelength predetermined into the flat waveguide. The coherent light propagates through the flat waveguide (2) with an evanescent field (6) that propagates along an outer surface (5) of the flat waveguide. The outer surface (5) of the flat waveguide comprises binding sites (7) therein capable of binding target samples (8) to the binding sites (7) so that the light from the evanescent field (6) is spread across the target samples (8) linked to the binding sites (7). The connection sites (7) are arranged along a plurality of predetermined lines (9) which are arranged so that the light diffuses from the constructively in a predetermined detection location with a difference in optical path length which is an integer multiple of the predetermined wavelength.
公开号:BR112014017474B1
申请号:R112014017474-1
申请日:2013-01-17
公开日:2020-11-10
发明作者:Christof Fattinger
申请人:F. Hoffmann-La Roche Ag；
IPC主号:

专利说明:

[0001] The present invention relates to a device for use in detecting binding affinities as well as a system and method for detecting binding affinities in accordance with the respective independent claim.
[0002] Such devices are used, for example, as biosensors in a wide variety of applications. A particular application is the detection or monitoring of process or connection affinities. For example, with the aid of such biosensors, several assays that detect the binding of target samples to binding sites can be performed. Typically, large numbers of such assays are performed on a biosensor at points that are arranged in a two-dimensional microarray on the surface of the biosensor. The use of microarrays provides a tool for the simultaneous detection of binding processes or affinities of different target samples in high-throughput screenings, in which large amounts of DNA, proteins or molecules similar to target samples can be analyzed quickly. To detect the affinities of target samples for binding to specific binding sites (for example, the affinities of target molecules for binding to different capture molecules), a large number of binding sites are immobilized on the surface of the biosensor at points that can be applied, for example, inkjet marking. Each point forms an individual measurement zone for a predetermined type of capture molecules. The affinity of a target sample to a specific type of capture molecule is detected and is used to provide information about the binding affinity of the target sample.
[0003] A known technique for detecting binding affinities of target samples uses labels capable of emitting fluorescent light upon excitation. For example, fluorescent markers can be used as labels to label target samples. Upon excitation, the fluorescent markers are forced to emit a fluorescent light that has a characteristic emission spectrum. The detection of this characteristic emission spectrum at a particular point indicates that the labeled target molecule has been linked to the particular type of binding sites present at the respective point.
[0004] A sensor to detect labeled target samples is described in the article "Zeptosens' protein microarrays: A novel high performance microarray platform for low abundance protein analysis", Proteomics 2002, 2, S. 383 to 393, Wiley-VCH Verlag GmbH , 69451 Weinheim, Germany. The sensor described in this document comprises a flat waveguide arranged on a substrate and a grid to couple a coherent light of a predetermined wavelength to the interior of the flat waveguide. An additional grid is arranged at that end of the flat waveguide away from the grid to couple the light inside the waveguide. The coherent light that has been propagated through the flat waveguide is coupled out of the waveguide by the additional grid. The out-coupled light is used to adjust the coherent light coupling of predetermined wavelength to the inside of the flat waveguide. Coherent light propagates through the flat waveguide under full reflection with an evanescent field of coherent light that propagates along the outer surface of the flat waveguide. The depth of penetration of the evanescent field into the lower refractive index medium on the outer surface of the flat waveguide is in the order of magnitude of a fraction of the wavelength of the coherent light that propagates through the flat waveguide. The evanescent field excites the fluorescent markers of the labeled target samples linked to the binding sites arranged on the surface of the flat waveguide. Due to the excessively small penetration of the evanescent field into the optically thinner medium on the outer surface of the flat waveguide, only labeled samples attached to the binding sites immobilized on the outer surface of the flat waveguide are excited. The fluorescent light emitted by these markers is then detected with the aid of a CCD camera.
[0005] Although it is essentially possible to detect binding affinities with the use of fluorescent labels, this set of procedures is disadvantageous in that the detected signal is produced by the labels instead of being produced by the binding partners themselves. In addition, labeling the target samples requires additional work steps. In addition, labeled target samples have a comparatively higher cost. Another disadvantage is the falsification of the results caused by the effects of sudden cooling or photobleaching.
[0006] It is an object of the present invention to provide a device for use in detecting binding affinities of a target sample as well as a system and method that can detect such binding affinities that overcome or at least greatly reduce the disadvantages of the sensor. prior art described above.
[0007] In accordance with the invention, this objective is achieved by a device for use in detecting binding affinities. The device comprises a flat waveguide disposed on a substrate and additionally comprises an optical coupler for coupling a coherent light of a predetermined wavelength within the flat waveguide so that the coherent light travels through the flat wave with an evanescent field of coherent light that propagates along an outer surface of the flat waveguide. The outer surface of the flat waveguide comprises binding sites therein capable of binding target samples to binding sites so that light from the evanescent field is scattered over the target samples bound to the binding sites. The binding sites are arranged along a plurality of predetermined lines, with the predetermined lines being arranged so that the light scattered by the target samples connected to the binding sites interferes with a predetermined detection location with a difference and length of optical path which is an integer multiple of the predetermined wavelength of light.
[0008] The detection of binding affinities according to the invention is not limited to specific types of target samples or to a type of binding sites, but rather the binding characteristics of molecules, proteins, DNA, etc. can be analyzed for any type of binding sites in the flat waveguide. The detection of binding affinities can be achieved in a label-free manner. Alternatively, scattering intensifiers (for example, scattering labels) that spread light heavily can be used to increase detection sensitivity. Such spreading intensifiers can be a nanoparticle (alone or with a binder) or in another example a colloidal particle. The binding characteristic to be analyzed can be of the static type (for example, it can be analyzed whether or not a target sample is linked to the binding sites) or of the dynamic type (for example, the dynamics of the binding process over time can be analyzed). Binding sites are locations on the outer surface of the flat waveguide to which a target sample can bind. For example, binding sites may comprise capture molecules that are immobilized on the outer surface of the flat waveguide, or they may simply comprise locations activated on the outer surface of the flat waveguide capable of binding target samples to activated locations, or they can be incorporated in any other way to link target samples to the desired locations on the outer surface of the flat waveguide. The plurality of predetermined lines may comprise individual separate lines or may comprise a line pattern in which the individual lines are connected to form a single line, for example, a sinuous single line pattern. The distance between adjacent predetermined lines along which the binding sites are arranged is chosen in relation to the predetermined wavelength of the light. Preferred distances between adjacent predetermined lines are in the order of more than 100 nm. A range of about 100 nm to about 1000 nm for the distance between adjacent predetermined lines is preferred for the use of visible light in the flat waveguide so that scattered light can be detected by standard optical means. In addition, it is preferred that the flat optical waveguide has a high refractive index in relation to the medium on the outer surface of the flat waveguide, so that the depth of penetration of the evanescent field is only small and the coherent light fraction that propagates in the evanescent field is high. For example, the refractive index of the flat waveguide can be in the range of 1.6 to 2.5, while the refractive index of the medium on the surface of the flat waveguide is typically in the range of 1 to 1.5. For example, the binding sites may comprise capture molecules that are immobilized on the outer surface of the flat waveguide. The immobilized capture molecules together with the target samples attached to them form a plurality of scattering centers that spread the coherent light from the evanescent field. The coherent light that travels along the flat waveguide has a predetermined wavelength and is preferably monochromatic (ideally in a single wavelength). Since the light from the evanescent field that propagates along the surface of the flat waveguide is coherent as well as the light that propagates within the plane waveguide, the coherent light from the evanescent field is spread consistently across the scattering centers. formed by the target molecules attached to the capture molecules (or, more generally, by the target sample attached to the binding sites) which are arranged on different predetermined lines. The light scattered at any location can be determined by adding the contributions from each of the individual scattering centers. A maximum limit of the scattered light is located at the predetermined detection location since the predetermined lines are arranged so that, at the predetermined detection location, the optical path length of the light scattered through the different scattering centers is different by a multiple integer. the wavelength of light. For a maximum signal at the detection location, the optical path length of the light from the optical coupler to the predetermined lines and from them to the predetermined detection location is also an integer multiple of the predetermined wavelength. Thus, the light scattered by the target samples linked to the binding sites interferes with a predetermined detection location. The requirement for constructive interference is satisfied by any scattered light that is added to the detectable signal at the detection location. The predetermined detection location is not limited to a particular shape, for example, it can be shaped like a tip or a strip. The arrangement of the connection sites "along the predetermined lines" represents the optimal case in which all the connection sites are exactly on the predetermined lines. Such an optimal arrangement of the binding sites results in maximum signal at the detection location. It is obvious to the person skilled in the art that in practice the arrangement of the connection sites can deviate somewhat from such an optimal arrangement. For example, the deviation can be caused by the method for arranging the connection sites on the outer surface of the flat waveguide, as will be explained in more detail below.
[0009] In accordance with an aspect of the device according to the invention, the distance between adjacent predetermined lines decreases in the direction of light propagation of the evanescent field. In general, the angles at which the scattered light from the evanescent field interferes with the predetermined detection location are different for the various scattering centers (target samples linked to the binding sites), which are arranged along the predetermined lines. Since at the predetermined detection location the scattered light must interfere to a maximum, the difference in the optical path length of the scattered light from the various scattering centers must be an integer multiple of the light's wavelength. The decrease in distance between adjacent predetermined lines takes this into account and causes the light to interfere to a maximum in the predetermined detection location, which does not need to be shaped like a point or a small point, but can still have , the format of a shoot or any other desired format.
[00010] According to a further aspect of the device according to the invention, the plurality of predetermined lines on which the connection sites are arranged comprises curved lines. The curvature of the lines is such that the light from the evanescent field scattered by the target samples connected to the binding sites arranged along these predetermined lines interferes to a maximum at the predetermined detection location. The detection location is preferably shaped like a tip. Each of the individual predetermined lines can have a curvature that is different from the curvature of the other predetermined lines. In practice, the detection location is not a tip but it can be a small dot or a strip that has a length that is less than the length of the predetermined lines along which the connection sites are arranged. The curvature of each individual curved predetermined line is chosen so that the optical path length of the light that propagates from the optical coupler to the individual predetermined line and hence the predetermined detection location is a multiple integer of the predetermined wavelength of the light from spread to the entire curved line. This is also advantageous because the light scattered by the scattering centers located in the outer sections of the predetermined lines contributes to the signal in the spatially reduced area of the tip-shaped detection location (either with a dot or strip shape).
[00011] In accordance, furthermore, with an additional aspect of the device according to the invention, the plurality of predetermined lines is arranged on the outer surface of the flat waveguide so that their locations in Xj.yj coordinates are geometrically defined by the equation
on what
[00012] At the wavelength in the vacuum of the propagation light,
[00013] N is the effective refractive index of the guided mode in the plane waveguide; N depends on the thickness and refractive index of the flat waveguide, the refractive index of the substrate, the refractive index of a medium on the outer surface of the flat waveguide and the polarization of the guided mode,
[00014] ns is the refractive index of the substrate,
[00015] f is the thickness of the substrate,
[00016] Ao is an integer that is chosen to be close to the product of the refractive index ns and the thickness f of the substrate divided by the wavelength À, and
[00017] j is a sequential integer that indicates the index of the respective line.
[00018] The integer chosen Ao assigns negative x values in the center of the lines with negative j values and positive x values in the center of the lines with positive j values. Or in other words, the integer Ao defines the origin of the x, y coordinate frame that is used to locate the lines on the outer surface of the plane waveguide; the chosen Ao value places the detection location at x = 0, y = 0, z = -f.
[00019] As already outlined above, for an improved signal at the predetermined detection location it is preferred that the plurality of predetermined lines be arranged so that the scattering centers arranged along these predetermined lines are located in a structure similar to the curved grid with decreasing distance between adjacent predetermined lines. Such an arrangement satisfies the condition that the difference in the optical path length for the light that propagates from the optical coupler to the individual predetermined lines and spread across the scattering centers to the predetermined detection location is an integer multiple of the predetermined wavelength of the light. that propagates in the waveguide. In addition, the optical path length of the light that propagates from the optical coupler to the individual predetermined lines and from there to the predetermined detection location is an integer multiple of the predetermined wavelength of the propagation light for the entire curved line. Thus, it is possible to form a compact device due to the connection sites that are arranged on the surface of the flat waveguide while the detection location can be formed on the surface of the lower substrate that bears the flat waveguide.
[00020] Two modalities are particularly envisaged as to how the binding sites can be arranged along the plurality of predetermined lines. According to a first embodiment, the binding sites comprise capture molecules attached to the plane waveguide surface along predetermined lines only. These capture molecules are capable of binding target samples and are immobilized on the outer surface of the flat waveguide (although, as mentioned above, the binding sites can be formed by the activated surface of the flat waveguide itself). The immobilization of the capture molecules on the outer surface of the flat waveguide along the predetermined lines can generally be carried out by any suitable method, for example, it can be carried out using photolithographic methods that use a lithographic mask with curved lines. It goes without saying that the arrangement of the binding sites along the predetermined lines must be understood in any embodiment of the invention in the sense that most of the binding sites - in the present embodiment, the capture molecules - are located along predetermined lines and does not explicitly include that some connection sites are arranged in locations other than those.
[00021] According to a second embodiment, the binding sites again comprise capture molecules that have the ability to bind target samples, which is not a restriction on a particular type of binding site or on a particular type of target sample. Again, capture molecules capable of binding target samples. However, the disposition of capture molecules capable of binding target molecules along predetermined lines is accomplished by distributing and immobilizing capture molecules capable of binding target samples on (all) the surface of the plane waveguide , and subsequently deactivating those capture molecules that are not arranged along the predetermined lines. The term "deactivate", in this sense, refers to any suitable method to alter the binding capacity of the capture molecules (for example, by exposing the capture molecules to light for a predetermined time) in order to achieve that they no longer have the ability to bind target samples. According to this embodiment of the invention, the capture molecules can be applied uniformly or statically to the outer surface of the flat waveguide. After the deactivation of the capture molecules that are arranged between the predetermined lines, only the capture molecules arranged along the predetermined lines (these have not been deactivated) capable of binding a target sample. However, the deactivated capture molecules remain immobilized on the outer surface of the flat waveguide.
[00022] This modality has the additional advantage that the contribution of the signal generated by the light scattered by the target molecules linked to the capture molecules to the general signal at the detection location is increased. Generally, the difference between the signals from the light scattered by the target molecules attached to the capture molecules and the light scattered by the capture molecules without any target molecules therein is small compared to the light scattered by the capture molecules alone. Assuming that the scattering properties of the capture molecules disposed along the predetermined lines (which have not been deactivated) and the deactivated capture molecules disposed between the predetermined lines are identical and additionally supposing that the capture molecules are evenly distributed over the outer surface of the flat waveguide, then ideally no signal is produced at the detection location after the capture molecules have been immobilized on the outer surface of the flat waveguide and after the capture molecules arranged between the lines have been disabled. In practice, however, the deactivation of the capture molecules slightly alters the spreading properties of the capture molecules, so it may not be ideal to deactivate all of the capture molecules that are arranged between the predetermined lines. Instead, only the vast majority of capture molecules arranged between predetermined lines can be deactivated. The capture molecules are deactivated to a certain degree so that the general signal at the detection location produced by those capture molecules arranged along the predetermined lines and by the deactivated ones and by the few non-deactivated capture molecules arranged between the lines predetermined values is at a minimum and is preferably zero. Assuming that the signal thus obtained at the detection location can be reduced to zero, this means that after adding the target samples the signal produced at the detection location results only from the target samples linked to the capture molecules. If no target sample is attached to the capture molecules, the signal at the detection location remains zero. This increases the detector's sensitivity to the signal generated by the light scattered by the target molecules attached to the capture molecules at the detection location.
[00023] In accordance with an additional aspect of the device according to the invention, the flat waveguide has a refractive index nw which is substantially higher than the refractive index ns of the substrate and which is still substantially higher that the refractive index nmed of the medium on the outer surface of the flat waveguide, so that, for a predetermined wavelength of light, the evanescent field has a penetration depth in the range of 40 nm to 200 nm. The term "substantially higher" should be understood as referring to a difference in refractive index that allows a coupling of the light inside the flat waveguide in which it propagates under total reflection. The light that travels along the flat waveguide has an evanescent field that travels along the outer surface of the flat waveguide. The evanescent field has a depth of penetration that depends on the nmed index, the effective refractive index N in the guided mode, as well as on the wavelength of the propagation light, so that the depth of penetration can be adapted so that the light from the field evanescent is spread consistently across target samples attached to the binding sites located on or near predetermined lines on the outer surface. The approximate values of the penetration depth mentioned above should be understood as explicitly including the exact limit values of the same.
[00024] In accordance with an additional aspect of the device according to the invention, the device comprises an additional optical coupler for coupling out the light that has been propagated through the plane waveguide. Both the optical coupler for coupling the light inside the flat waveguide as well as the additional optical coupler for coupling out the light that has been propagated through the flat waveguide can comprise an optical grid for coherently coupling the light inward and out of the flat waveguide. The optical coupler and the additional optical coupler comprise an optical grid for coherently coupling the light evenly in and out of the plane waveguide under an respective predetermined internal coupling angle or external coupling angle. The angle of internal coupling or angle of external coupling is determined by the wavelength of the light as well as the characteristic of the optical coupler. However, within the scope of the invention, light can also be coupled in and out of the flat waveguide by any other suitable means for coupling light in and out of a flat waveguide of a thickness in the range of some nanometers to a few hundred nanometers. Just as an example, an alternative optical coupler can be an optical prism.
[00025] In accordance with an additional aspect of the device of the invention, the flat waveguide has a first end section and a second end section which are arranged at opposite ends of the flat waveguide with respect to the direction of light propagation. through the flat waveguide. The first end section and the second end section comprise a material capable of absorbing the wavelength of light that propagates through the flat waveguide. The absorptive material minimizes the reflections of light that travel along the flat waveguide towards the respective end section and back into the flat waveguide. This enhances the detected signal as the light that has been reflected from the ends of the flat waveguide is eliminated or at least considerably minimized.
[00026] In accordance with an additional aspect of the device according to the invention, a plurality of measurement zones are arranged on the outer surface of the flat waveguide. In each measurement zone, the connection sites are arranged along the plurality of predetermined lines. For high-throughput screening, the simultaneous binding affinity detections of a sample can be achieved for different types of binding sites and target samples by having the respective target samples linked to the binding sites in separate measurement zones. Each measurement zone has a corresponding individual detection location to allow separate detection of scattered light from the evanescent field.
[00027] In accordance with an additional aspect of the device according to the invention, the plurality of measurement zones comprises measurement zones of different sizes. All sizes of the measurement zones are known. At the respective detection location, the light scattered over corresponding measurement zones of different size in which the same type of target samples are linked to the same type of binding sites can be compared. The intensity of the light scattered at the detection location has a quadratic correlation with the number of scattering centers in the respective measurement zone on the flat surface of the waveguide. Thus, for uniform distribution and area density of scattering centers in measurement zones of different sizes, the intensities of the light scattered at the respective detection locations of corresponding measurement zones of different sizes have a quadratic correlation with the size of the zones of measurement. respective measurement. Therefore, the intensities of the scattered light in detection locations of measurement zones of different sizes can be used to verify that the measured intensities are in fact representative of the light scattered by the scattering centers arranged in the predetermined lines.
[00028] According to one aspect of the invention, each measurement zone has an area greater than 25 pm2, in which the plurality of predetermined lines has a distance between adjacent predetermined lines less than 1.5 pm, in particular less than 1 pm. This makes it possible to achieve highly integrated devices with high numbers of measurement zones, ie 1,000, 10,000, 100,000, ..., up to 4 X 106 measurement zones per square centimeter.
[00029] Advantageously, the connection sites are arranged along at least two pluralities of predetermined lines in a single measurement zone. Each of the two pluralities of predetermined lines is arranged so that the light scattered by the target samples connected to the binding sites arranged along the respective plurality of predetermined lines interferes with a difference in optical path length which is an integer multiple of the predetermined wavelength of light at an individual detection location for each of the plurality of predetermined lines. The individual detection locations are spatially separated from each other. More than a plurality of predetermined lines in the measurement zone that are arranged to provide spatially separate detection locations allow additional methods to be detected for connection events (for example, the detection of cooperative connections or the detection of cascades) reaction).
[00030] In accordance with another aspect of the device according to the invention, the device comprises a diaphragm that has an opening that is arranged so that the light at the detection location is allowed to pass through the opening while the light at a location different from the detection location it is blocked by the diaphragm. A mechanical diaphragm as well as an electronic diaphragm can be adapted to block all light beyond that which is scattered to the detection location. Advantageously, the diaphragm can be formed on the outer surface of the substrate on that side away from the flat waveguide. For example, a non-transparent material, for example, a chrome layer, can be applied to the substrate surface away from the waveguide. The non-transparent chromium layer has a transparent opening at the detection location through which the light scattered to the detection location can pass while the rest of the light not spread into the opening is blocked.
[00031] In accordance with an even further aspect of the device according to the invention, the diaphragm additionally comprises at least one additional opening which is arranged adjacent to the opening when viewed in the direction of light propagation through the plane waveguide. The additional opening is located adjacent to the opening so that an inconsistent light scattered to the additional opening can pass through the additional opening. Advantageously, the detected inconsistent background light can be corrected by using a diaphragm that has an additional aperture. The additional aperture does not, by itself, detect the inconsistent background light at the detection location, but allows the determination of the amount of inconsistent light at the detection location from a measurement of the inconsistent light at a location other than the detection location . The amount of inconsistent light thus determined at the detection location cannot be separated from the light at the detection location, but can be subtracted from every signal at the detection location, once every signal at the detection location has been measured by a detector. For improved correction, an additional first opening is located in relation to the direction of the propagation light in front of the detection location and a second additional opening is located behind the detection location. Such a configuration allows to detect an average value for the inconsistent light at the detection location to correct the signal at the detection location.
[00032] Another aspect of the invention relates to a system for detecting binding affinities which comprises a device for detecting binding affinities according to the invention. The system additionally comprises a light source for emitting coherent light of a predetermined wavelength, the light source and the device being arranged in relation to each other so that the coherent light is coupled within the guide. plane wave through the optical coupler. Alternatively, the system additionally comprises optical means for scanning and / or adjusting the light angle that falls on the optical coupler since the exact coupling angle of the optical coupler can vary from device to device. Alternatively, the wavelength of the light emitted by the light source in the system can be adjusted, which can be advantageous if the angle of the light that falls on the optical coupler is fixed for construction reasons.
[00033] In accordance with yet another aspect of the system according to the invention, the system additionally comprises an optical imaging unit, the optical imaging unit being focused in order to produce an image of the detection location of the device . The optical imaging unit has the ability to provide an image of the predetermined detection location in which the light scattered by the target samples attached to the binding sites interferes with a difference in optical path length which is an integer multiple of the wavelength predetermined amount of light. The optical imaging unit can be used to image the light present at the detection location to an observation location. The optical imaging unit can be adapted to image both the light from the detection location as well as the light from the additional opening or additional openings, as this light can be used to subtract the inconsistent background light from the entire the light present at the detection location. Alternatively or in addition, the optical imaging unit can be used to select only the light at the detection location by focusing the optical imaging unit on the detection location. Thus, a diaphragm is no longer needed.
[00034] Another aspect of the invention relates to a method for detecting binding affinities. The method comprises the steps of:
[00035] - provide a device that comprises a flat waveguide arranged on a substrate and an optical coupler,
[00036] - couple the coherent light of a wavelength pre-terminated inside the flat waveguide so that the coherent light propagates along the flat waveguide with an evanescent field of coherent light that propagates along an outer surface of the flat waveguide,
[00037] - attaching the target samples to the binding sites arranged along a plurality of predetermined lines on the outer surface of the flat waveguide,
[00038] - detecting, in a predetermined detection location, the light from the evanescent field scattered by the target samples connected to the binding sites arranged along the predetermined lines, with the light scattered by the target samples connected to the binding sites has, at the predetermined detection location, a difference in optical path length which is an integer multiple of the predetermined wavelength of light.
[00039] Additional advantageous aspects of the invention become apparent from the following description of the modalities of the invention with reference to the accompanying drawings in which:
[00040] Figure 1 shows a perspective view of an embodiment of the device according to the invention;
[00041] Figure 2 shows a sectional view of the device of Figure 1;
[00042] Figure 3 shows an illustration of different optical paths for the light of the evanescent field that propagates along the external surface and that is spread to the detection location;
[00043] Figure 4 shows a measurement zone of the device according to the invention comprising an arrangement of a plurality of predetermined lines, the connection sites being immobilized along the predetermined lines;
[00044] Figure 5 shows the measurement in Figure 4, with some target samples being linked to the binding sites;
[00045] Figure 6 shows a measurement zone of a device according to the invention that comprises an arrangement of a plurality of predetermined lines, the connection sites being immobilized along the predetermined lines and between the predetermined lines;
[00046] Figure 7 shows the measurement zone of Figure 6, with those connection sites arranged between the predetermined lines being deactivated;
[00047] Figure 8 shows the measurement zone of Figure 7 with the target samples are added;
[00048] Figure 9 shows the measurement zone of Figure 8 with the target samples being linked to the immobilized binding sites along the predetermined lines;
[00049] Figure 10 shows an illustration of the construction of a void section in which the predetermined lines of a measurement zone must be eliminated;
[00050] Figure 11 shows the measurement zone of Figure 10, with a void section in which the predetermined lines are eliminated;
[00051] Figure 12 shows a top view of an additional embodiment of the device according to the invention comprising a plurality of measurement zones;
[00052] Figure 13 shows a bottom view of the device of Figure 10, with one opening provided at the detection location and two additional openings provided at locations in front of and behind the detection location for each measurement zone. ;
[00053] Figures 14 to 17 show a portion of a measurement zone of a device according to the invention at different stages of a target sample connection process;
[00054] Figure 18 shows a sectional view of an additional embodiment of the device in which the device comprises an additional carrier substrate;
[00055] Figure 19 shows an illustration of different optical paths for the light of the evanescent field spread over two different pluralities of predetermined lines arranged in a single measurement zone;
[00056] Figure 20 shows a top view of the device of Figure 18 which has twelve measurement zones arranged in it, with three pluralities of predetermined lines being arranged in each measurement zone; and
[00057] Figure 21 shows a bottom view of the device of Figure 18 with openings in a non-transparent layer formed on top of the additional carrier substrate.
[00058] Figure 1 shows a perspective view of an embodiment of the device according to the invention for detecting binding affinities in a sample. The device comprises a substrate 3 of a transparent material which, in the embodiment shown, is shaped like a rectangular cube, without being limited to that shape. A flat waveguide 2 (see also Figure 2) is arranged on the upper side of the substrate 3, within which a coherent light 1 is coupled so that the coherent light propagates through the flat waveguide 2 under total reflection . Since the plane waveguide 2 has a thickness in the range of a few nanometers to a few hundred nanometers only, it is not illustrated as a separate layer in Figure 1, but is shown exaggerated in Figure 2. As illustrated by the arrows parallel in Figure 1, the coherent light 1 of a predetermined wavelength is coupled through the substrate 3 inside the plane waveguide 2 with the aid of a grid 4 that acts as an optical coupler. The coherent light coupled inside the flat waveguide 2 propagates along the flat waveguide 2 with an evanescent field 6 (represented by an arrow) that penetrates in the middle above the upper surface of the flat waveguide 2 (see again Figure 2). The evanescent field 6 propagates along the outer surface 5 of the flat waveguide 2. A measurement zone 10 arranged on the outer surface of the flat waveguide 2 comprises a plurality of predetermined lines 9 (each of the lines shown represents a multiplicity of lines, in particular fifty lines in the present example of such a device, and only one of such a measurement zone is shown in Figure 1 for the sake of clarity). The binding sites (not shown in Figure 1) to which the target samples can bind are arranged along these predetermined lines 9. The coherent light from the evanescent field 6 is spread across the target samples linked to the binding sites within the zone measurement 10. Part of the light scattered by the target samples connected to the binding sites is directed to a detection location where the diaphragm 11 comprising an aperture 21 is arranged. The diaphragm 11 is produced from a non-transparent material and can, for example, be a layer of chromium which is applied to the lower surface of the substrate 3.
[00059] Figure 2 shows a sectional view of the device of Figure 1, the thickness of the flat waveguide being shown exaggerated for the purpose of explaining the general working principle. As can be seen, the light coupled inside the flat waveguide 2 with the aid of optical grid 4 propagates through the flat waveguide 2 under total reflection until it reaches an additional grid 13 arranged at the opposite end of the waveguide This additional grid serves as an additional optical coupler to couple the light out of the flat waveguide. To avoid reflections and to minimize incoherent background light, a first end section 14 and a second end section 15 of the flat waveguide 2 comprise a material with absorbency. Corresponding to the light propagating in the flat waveguide, the evanescent field 6 propagates along the outer surface 5 of the flat waveguide 2.
[00060] The refractive index nw of the flat waveguide 2 is substantially higher than the refractive index ns of the substrate 3 and also substantially higher than the refractive index nmed of the medium on the outer surface 5 of the flat waveguide 2. The refractive index nmed of the medium on the outer surface 5 can vary depending on the type of sample applied to it. For example, the refractive index nmed for the medium on the outer surface 5 may be of the order of the refractive index of water in case the target sample is present in an aqueous solution applied to the outer surface 5 of the flat waveguide 2, or it may be the order of the refractive index of air in the case of dry target samples, or it can be of the order of the refractive index of a hydrogel layer 16 in the case of the binding sites to which the target samples 8 can be attached are contained in a layer of hydrogel 16 on the outer surface 5. The depth of penetration of the evanescent field 6 in the middle on the outer surface 5 of the flat waveguide 2 (distance between the outer surface 5 of the flat waveguide 2 and the declining intensity of 1 / e2 of the evanescent field 6) depends on the nmed index of the medium on the outer surface 5 of the flat waveguide 2, the effective refractive index N of the guided mode and the wavelength À of the light.
[00061] The light in the evanescent field 6 that spreads along the outer surface 5 of the flat waveguide 2 is scattered by the target samples 8 connected to the binding sites, and these binding sites can comprise the capture molecules 7 able to connect the target samples 8 and which are arranged in the measurement zone 10 along the predetermined lines 9 (Figure 1). In Figure 2, it is indicated by arrows of decreasing length that the distance between adjacent predetermined lines along which the capture molecules 7 are arranged decreases when viewed in the direction of light propagation. As can be seen further, in the modality shown in Figure 2 the target samples 8 were applied to the measurement zone by distributing a drop containing the target samples 8. Part of the light scattered by the target samples 8 linked to the capture molecules 7 is directed to the detection location where opening 21 of diaphragm 11 is arranged. As an option, the light at the detection location can be imaged on a photodetector 20 by an optical imaging unit 19. Optical imaging unit 19 and photodetector 20 are shown surrounded by a box drawn in dashed lines, as they can be provided alternatively or in combination, and can in particular be supplied in combination in one unit.
[00062] Although it is already evident from Figure 2 that the lengths of the optical paths of the evanescent field light 6 that is spread over the target samples 8 linked to different capture molecules 7 at the detection location is different, this becomes even more more evident when looking at Figure 3 in which several of such different optical paths are explicitly shown. Part of the coherent light from the evanescent field 6 is scattered over the target samples 8 linked to different capture molecules 7 in order to interfere with the detection location which is the location of aperture 21 of the diaphragm 11. For a predetermined detection location, the dis - position and geometry of the predetermined lines 9 as well as the thickness of the substrate 3 are selected so that at the detection location the difference in the optical path length is an integer multiple of the predetermined wavelength of the coherent light. Thus, the interference of light at the detection location is the coherent additive overlap of the light scattered to the detection location by the target molecules 8 linked to the different capture molecules 7.
[00063] For the embodiment shown in Figure 3, the plurality of curved predetermined lines 9 is arranged on the outer surface 5 of the flat waveguide so that their locations on the plane of the outer surface 5 of the flat waveguide are geometrically expressed in Xj.yj coordinates by the equation
on what
[00064] À is the vacuum wavelength of the propagation light,
[00065] N is the effective refractive index of the guided mode in the plane waveguide; N depends on the thickness and refractive index of the flat waveguide, the refractive index of the substrate, the refractive index of a medium on the outer surface of the flat waveguide and the polarization of the guided mode,
[00066] ns is the refractive index of the substrate,
[00067] f is the thickness of the substrate,
[00068] Ao is an integer that is chosen to be close to the product of the refractive index ns and the thickness f of the substrate divided by the wavelength À, and
[00069] j is a sequential integer that indicates the index of the respective line.
[00070] Figure 4 shows a measurement zone 10 in an enlarged view comprising the predetermined lines 9 and the binding sites represented by the capture molecules 7 that are immobilized on the outer surface of the plane waveguide 5 (see Figure 1 ) along the predetermined lines 9. The immobilization of the capture molecules 7 along the predetermined lines can be performed only with the aid of sets of lithographic procedures, as discussed above. In Figure 5, target samples 8 are linked to some of the capture molecules 7. Since capture molecules 7 are arranged along the plurality of predetermined lines 9, target samples 8 linked to capture molecules 7 are also arranged along the plurality of predetermined lines 9. At the detection location this results in a coherent additive overlap of the light scattered by the scattering centers formed by the target samples 8 linked to the capture molecules 7, as explained above.
[00071] Figure 6, Figure 7, Figure 8 and Figure 9 show, again, a measurement zone 10 in an enlarged view. However, the way in which capture molecules 7 capable of binding target samples 8 have been immobilized along predetermined lines 9 is different.
[00072] As can be seen in Figure 6, in a first stage the capture molecules 7 are immobilized on (all) the outer surface of the flat waveguide in the measurement zone 10, so that there is no disposition of the molecules of capture along the plurality of predetermined lines 9. Thus, the light from the evanescent field spread by the capture molecules 7 does not interfere with the detection location in the manner described above.
[00073] As can be seen in Figure 7, the capture molecules arranged between the predetermined lines 9 have been deactivated so that no target sample can bind to these deactivated capture molecules 12. Consequently, the only capture molecules 7 capable of binding target samples are arranged along the plurality of predetermined lines 9. The precision of the immobilization of the capture molecules 7 along the predetermined lines 9 depends on the method of fixing, immobilizing or deactivating the capture molecules 7. As a result , the location of the immobilized capture molecules 7 capable of binding target samples 8 may not be exactly "on" predetermined lines 9, but may in some way be deviated from the exact location "on" predetermined lines 9. In practice, the deviation of the exact location "on" predetermined lines can be covered by a range that is less than a quarter of the distance from adjacent predetermined lines 9. This results in still constructive interference from scattered light to the detection location.
[00074] As will be explained in the introductory part, the deactivation of the capture molecules 12 arranged between the predetermined lines 9 is carried out so that after deactivation the general signal at the detection location (no target sample 8 has been added yet) produced by capture molecules deactivated 12 and capture molecules 7 capable of binding target samples 8 is set or adjusted to a minimum signal tuned to the detection location, ideally to zero.
[00075] The next step is to add target samples 8 to measurement zone 10 on the outer surface of the flat waveguide, as shown in Figure 8. Since only capture molecules 7 arranged along predetermined lines 9 capable of turn on target samples 8, target samples 8 are linked to those capture molecules 7 along the predetermined lines 9, as shown in Figure 9. Due to the signal tuned to the detection location caused by the disabled capture molecules 12 and the molecules capture 7 that have been set to a minimum previously (see above), the signal at the detection location is then mainly (or entirely, if the signal produced by the deactivated capture molecules and the capture molecules has been reduced to zero previously ) caused by the light scattered by the target samples 8 linked to the capture molecules 7 arranged along the predetermined lines.
[00076] Figure 10 shows a portion of the measurement zone 10 as described above to illustrate the construction of a void section in which the predetermined lines 9 must be eliminated to avoid second order Bragg reflections in the plane waveguide. Bragg reflections should be avoided as they result in a reduction in the intensity of the light that spreads along the flat waveguide. This is particularly disadvantageous if a plurality of measurement zones 10 are arranged one after the other on the outer surface of the flat waveguide in the direction of the propagation light. Thus, a decrease in the intensity of the spreading light spread over the subsequent measurement zones is not only due to the scattering processes described in the various measurement zones, but in addition it decreases due to the Bragg reflections in the plane waveguide. Since in each measurement zone the predetermined lines 9 in a circular section of the measurement zone have a distance between adjacent lines that satisfies the condition for the second order Bragg reflex, the second order Bragg reflex in the guide plane waveform defines an additional location 22 in which Bragg's reflected light constructively interferes. In the example shown, the points of intersection of the arc of circle 21 shown with the predetermined lines 9 indicate those locations of the predetermined lines 9 for which the Bragg condition is exactly satisfied, so that the light is reflected back and interferes so constructive in the additional location 22. This reflected light is not available for scattering in subsequently arranged measurement zones 10.
[00077] Figure 11 shows a measurement zone 10 comprising a region 23 in the vicinity of arc of circle 21 in which predetermined lines 9 are eliminated to avoid such second order Bragg reflections.
[00078] Figure 12 and Figure 13 show top and bottom views of an additional embodiment of the device according to the invention. This embodiment of the device comprises a plurality of measurement zones 10 of a first size and measurement zones 17 of a different size. Each measurement zone 10 comprises a region 23 in which the plurality of predetermined lines 9 are eliminated to avoid Bragg reflections (see above). In general, it is also possible that the measurement zones do not comprise regions 23. An optical grid 4 for coupling light inside the flat waveguide and an additional grid 13 for coupling the light outside the flat waveguide are provided. Between the optical grid 4 and the additional optical grid 13, a plurality of measurement zones 10 of the first size and measurement zones 17 of different size are arranged where the connection sites are arranged along the predetermined lines 9, as discussed in details above. The plurality of measurement zones 10 of the first size and the plurality of measurement zones 17 of different size allows simultaneous detection of different combinations of target samples and binding sites, so that a plurality of combinations of target samples and sites binding can be analyzed simultaneously for the binding affinity of specific target samples to specific binding sites. Alternatively, redundant measurements can be performed for the same combinations of target samples and binding sites.
[00079] From the bottom view of Figure 13 it is possible to observe that an opening 21 is provided for each measurement zone at the detection location, in which the scattered light has a difference in optical path length which is an integer multiple of the wavelength of the light that propagates in the waveguide to the scattering center in a predetermined line and from there to the predetermined detection location, also as discussed in detail above. It goes without saying that an optical imaging unit can be provided as discussed in detail in relation to Figure 2.
[00080] Measurement zones 17 of different size are arranged between measurement zones 10. Measurement zones 17 have a known size different from the size of measurement zone 10, all sizes being known. At the respective detection location, the light scattered over the measurement zones 10 and the corresponding measurement zones 17 can be compared (for the same type of target sample linked to the same type of connection sites). The intensity of the light scattered at the detection location has a quadratic correlation with the number of scattering centers in the measurement zone on the flat surface of the waveguide. Assuming that a uniform distribution and area density of scattering centers in measurement zones of different sizes, the intensities of the scattered light at the respective detection locations of the corresponding measurement zones of different sizes have a quadratic correlation with the size of the zones. measuring instruments. Consequently, the intensities of the scattered light at detection locations of the measurement zones of different sizes can be used to verify that the measured intensities are in fact representative of the scattered light by the scattering centers arranged in the predetermined lines.
[00081] For improved detection of binding affinities, two additional openings 18 are formed in substrate 3 in front of and behind each opening 21 dedicated to the respective measurement zone 10. Since the coherent light that propagates through the waveguide plane 2 can also be spread coherently along the path through the plane 2 waveguide, a contribution of this coherently scattered light is also detected at the detection location through aperture 21. Openings 18 in front of and behind the opening 21 at a predetermined distance the determination of an average signal representative of this scattered light in a coherent way that can be used to correct the signal detected at the detection location by subtracting the average signal from the inconsistent light from the general signal detected at the detection location. This correction of the signal at the detection location is particularly advantageous in combination with the aforementioned reduction in the background signal caused by scattering at the binding sites on any target molecules attached to them.
[00082] Figure 14 to Figure 17 show a portion of a measurement zone that is formed on the outer surface 5 of a flat waveguide according to the invention. Different stages of a process of binding target samples 8 to capture molecules 7 are shown. In this process, the binding of target samples 8 to capture molecules 7 is enhanced. Capture molecules 7 are immobilized on the outer surface 5. Subsequently, target samples 8 and ligands 24 are applied. The applied target samples 8 are allowed to bind to the capture molecules 7 until an equilibrium condition is reached in which the binding of the target samples 8 to the capture molecules 7 and the release of the target samples 8 from the molecules catches are in balance. The ligand is then activated (for example, by light) to reinforce the bonds between target samples 8 and capture molecules 7. Subsequently, unbound target samples 8 as well as unused ligands 24 will be removed per wash. Due to the reinforced bonds between the target samples 8 and the capture molecules 7 caused by the binders 24, the inadvertent washing out of the target samples 8 bound to the capture molecules 7 is avoided or at least considerably reduced. Thus, the signal at the detection location can be further enhanced. An example for such a process using photoactivated binders is described in detail in "Capture Compound Mass Spectrometry: A Technology for the Investigation of Small Molecule Protein Interactions", ASSAY and Drug Development Technologies, Volume 5, Number 3, 2007.
[00083] Figure 18 shows a cross-sectional view of a device which is essentially shown in Figure 1 but which in accordance with a modality additionally has a layer structure to be used, for example, in highly integrated systems (ie up to about 4x106 measurement zones per cm2). In the example shown, measurement zone 10 has an area of a size of about 25 pm2. This size makes it possible to arrange a multiplicity of measurement zones 10 on the outer surface 5 of the flat waveguide 2 to perform a multiplicity of measurements with the use of a single device. A reduced size measurement zone 10 is achieved, for example, by "virtually cutting" said reduced size area of 25 pm2 from a larger measurement zone. However, keeping the distance between the predetermined lines in such a small size measurement zone 10 unchanged would result in the fact that the cone formed by the scattered light in the target samples connected to the binding sites in the small size measurement zone 10 would have an angle opening that is substantially smaller than that of the oversized measurement zone. The smaller opening angle of the light cone would result in the fact that the same optical detection unit (comparable to Figure 2) that has been used to measure the larger measurement zone and that has a certain opening angle will measure not only light at the detection location, but also part of the incoherent background light. This worsens the signal-to-noise ratio (S / R ratio). To avoid this worsening of the S / R ratio, the distance between the measurement zone 10 and the detection location should be reduced ideally so that the angle of opening of the cone formed by the light scattered by the target samples connected to the connection sites of the measurement zone 10 of small size and which interferes with the detection location is identical to the opening angle of the optical detection unit. In order to reduce the distance between the reduced size measurement zone 10 and the detection location, the arrangement of the plurality of predetermined lines in the reduced size measurement zone 10 must be determined according to the formula described above with reference to Figure 3 of so that the light scattered by the target samples linked to the binding sites interferes with a new detection location. Since the distance between the small measurement zone 10 and the new detection location is only in the range of ten micrometers (pm) to a few hundred micrometers (pm), the thickness of substrate 3 can become impractical thin. In particular under laboratory conditions, it may be disadvantageous to manipulate devices that comprise substrates 3 that have a thickness in the range of 10 to a few hundred micrometers (pm). In order to improve the handling of such a device, the device according to this modality has the following layer structure (from bottom to top): an additional carrier substrate 24, a layer 111 of non-transparent material, the substrate 3 and the flat waveguide 2. The additional carrier substrate 24 is produced from a transparent material (eg glass, plastic) and has a thickness that makes the device suitable for handling (eg up to 3 mm) . The layer 111 of non-transparent material is formed on top of the additional carrier substrate 24. The layer 111 of non-transparent material is, for example, a layer of black chrome in which the openings 21, 18 are formed lithographically. The substrate is made of a transparent material and has a thickness that corresponds to the distance between the reduced size measurement zone 10 and the detection location. The flat waveguide 2 and the measurement zones 10 are in principle similar, as further described. Each measurement zone 10 can comprise more than a plurality of predetermined lines, as will be discussed in connection with Figure 19 below.
[00084] The illustration of the optical paths in Figure 19 is similar to Figure 3. However, two different pluralities of predetermined lines 9, 91 are arranged in a single measurement zone, and in each such zone the light is mirrored to different locations of spatially separated detection (foci) by the target samples linked to the different pluralities of predetermined lines 9, 91. The light from the evanescent field 6 that spreads along the outer surface 5 is mirrored in the target samples connected to the binding sites along the first plurality of predetermined lines 9 so as to interfere with the right side focus (bold lines) and target samples linked to the binding sites along the second plurality of predetermined lines 91 in order to interfere with the left side focus (lines dashed lines). This principle applies for each plurality of predetermined lines 9, 91 in relation to the respective detection location, so that additional pluralities of predetermined lines can be added within such a measurement zone (for example, three, as shown in Figure 20) . A sample that has the ability to link to the binding sites arranged on the two predetermined lines 9, 91 (Figure 19) can form cooperative links through multiple link interaction at the intersection of lines 9, 91. Such link interaction multiple has a high resistance. The two connections can be formed simultaneously or sequentially within short periods of time (instantaneously). Such multi-link interactions are optically detected at two separate detection locations that provide correlated signals at the two detection locations.
[00085] Figure 20 shows a top view of the device of Figure 18 with twelve measurement zones 10 arranged on the outer surface of the flat waveguide. In each measurement zone 10, three pluralities of predetermined lines are provided, and the target samples attached to the binding sites along these three pluralities of predetermined lines spread the coupled light inside the flat waveguide through the optical coupler 4 to the three spatially separate individual detection locations. The arrangement of three pluralities is advantageous since process cascades are detectable. Such a process cascade exists, for example, when a target sample is divided into separate products into the first type of capture molecule arranged to provide a signal at the first detection location. A first product of this reaction thus does not bind to the second type of capture molecules in order to provide a signal at the second detection location. A second reaction product binds to the third type of capture molecule to provide a signal at the third detection location.
[00086] Figure 21 shows a bottom view of the device of Figure 20. From below, through the additional transparent carrier substrate 24, layer 111 of the non-transparent material disposed on top of the additional carrier substrate 24 can be seen. Groups of nine openings are formed in the non-transparent material layer 111. Structurally, the non-transparent material layer 111 comprises several openings that are shaped to block any light apart from the scattered light required for measurement at the respective detection location. For optimal suppression of diffuse non-coherent background light at the detection location, the diameter of a round aperture is chosen to be larger than the diameter of the focal point produced by scattered light that interferes with the detection location. At first, the size is provided by the Abbe formula for calculating the theoretically possible resolution of the microscope:
[00087] do = À / 2ns signal = = Àf / ns D where
[00088] À is the vacuum wavelength of the coherent light that propagates in the flat waveguide,
[00089] α is half the opening angle of the measurement zone,
[00090] ns is the refractive index of substrate 3,
[00091] f is the focal length of the measurement zone, and
[00092] D is the diameter of the measurement zone.
[00093] Additional openings are formed in the non-transparent layers 111 in front of and behind the openings 21 (see Figure 18) to determine a medium background signal. The shape of the openings can be chosen to correspond to the shape of the focal point formed by the light that interferes with the detection location. It may be advantageous to provide an elongated opening 21 (which extends in the direction of propagation of the evanescent field) in order to avoid cutting the light to be detected at the detection location with the opening edge, for example, in the case of changes in location of the focal point caused by changes in the refractive index of the sample applied to the outer surface of the flat waveguide or caused by small changes in the thickness of the flat waveguide.
[00094] Although the modalities of the invention have been described with the aid of the drawings, several modifications and alterations of the described modalities are possible, without departing from the general teaching underlying the invention. Therefore, the invention should not be understood as being limited to the described modalities, but instead the scope of protection is defined by the claims.

权利要求:
Claims (18)
[0001]
1. Device for use in detecting binding affinities, the device being characterized by the fact that it comprises a flat waveguide (2) arranged on a substrate (3) and having an optical coupler (4) to couple coherent light ( 1) of a predetermined wavelength to the flat waveguide (2) so that the coherent light propagates through the flat waveguide (2) with an evanescent field (6) of the coherent light that propagates along a outer surface (5) of the flat waveguide (2), where the outer surface (5) of the flat waveguide (2) comprises binding sites (7) therein capable of binding the target samples (8) to the binding sites (7) so that the light from the evanescent field (6) is spread over the target samples (8) linked to the binding sites (7), where the binding sites (7) are arranged over a plurality of predetermined lines (9), the predetermined lines (9) being arranged so that the light scattered by the target samples (8) linked to the binding sites (7) interfere with a predetermined detection location with a difference in optical path length which is an integer multiple of the predetermined wavelength of the coherent light.
[0002]
2. Device according to claim 1, characterized by the fact that the distance between adjacent predetermined lines (9) decreases in the direction of light propagation of the evanescent field.
[0003]
3. Device according to claim 1, characterized by the fact that the plurality of predetermined lines (9) in which the connection sites (7) are arranged comprise curved lines, with the curvature of the lines being such that the light of the evanescent field (6) spread by the target samples (8) linked to the binding sites (7) interfere with a predetermined detection point such as a detection location.
[0004]
4. Device according to claim 1, characterized by the fact that the plurality of predetermined lines (9) are arranged on the outer surface (5) of the flat waveguide (2) so that their locations are geometrically defined by the equation
[0005]
5. Device according to claim 1, characterized by the fact that the binding sites comprise capture molecules (7) attached to the surface of the flat waveguide (2) along the predetermined lines (9) only, being capture molecules capable of binding target samples (8).
[0006]
6. Device according to claim 1, characterized by the fact that the binding sites comprise capture molecules (7) capable of binding target samples (8), while capture molecules (7) capable of binding target samples (8) are arranged along predetermined lines (9) distributing capture molecules (7) capable of binding target samples (8) on the outer surface (5) of the flat waveguide (2) and deactivating those capture molecules (12) that are not arranged along predetermined lines (9).
[0007]
7. Device according to claim 1, characterized by the fact that the flat waveguide (2) has a refractive index (nw) that is substantially higher than the refractive index (ns) of the substrate (3) and that is , still substantially higher than the refractive index (nmed) of the medium on the outer surface (5) of the flat waveguide (2), so that for a predetermined wavelength of light the evanescent field (6) has a depth of penetration in the range of 50 nm to 200 nm.
[0008]
8. Device according to claim 1, the device being characterized by the fact that it comprises an additional optical coupler (13) for coupling out the coherent light that has been propagated through the plane waveguide (2), as well as the optical coupler (4) for coupling out the coherent light that has been propagated through the flat waveguide (2) comprise an optical grid (4, 13) for coherently coupling the coherent light in and out of the flat waveguide (2).
[0009]
9. Device according to claim 1, characterized by the fact that the flat waveguide (2) has a first end section (14) and a second end section (15) which are arranged at opposite ends of the plane wave (2) in relation to the direction of light propagation through the plane wave guide (2), the first end section (14) and the second end section (15) each comprising a material with absorption capacity in the wavelength of the light that propagates through the flat waveguide (2).
[0010]
10. Device according to claim 1, characterized by the fact that a plurality of measurement zones (10, 17) is arranged on the outer surface (5) of the flat waveguide (2), in which in each measurement zone (10) the binding sites (7) are arranged along the plurality of predetermined lines (9).
[0011]
11. Device according to claim 10, characterized by the fact that the plurality of measurement zones comprise measurement zones of different sizes (10, 17).
[0012]
12. Device according to claim 10, characterized by the fact that each measurement zone (10) has an area greater than 25 pm2, and in which the plurality of predetermined lines (9) has a distance between adjacent predetermined lines ( 9) less than 1.5 pm.
[0013]
13. Device according to claim 10, characterized by the fact that the connection sites (7) are arranged along at least two pluralities of predetermined lines (9) in a single measurement zone (10), each of which one of the two pluralities of predetermined lines (9) is arranged so that the coherent light of the evanescent field that is spread by the target samples (8) connected to the connection sites (7) arranged along the respective plurality of the at least two pluralities predetermined lines (9) interfere with an individual predetermined detection location for each of the respective plurality of predetermined lines (9) with a difference in the length of the optical path which is an integer multiple of the predetermined wavelength of the light coherent with the predetermined individual detection locations for each of the respective pluralities of predetermined lines (9) being spatially separated from each other.
[0014]
14. Device according to claim 10, characterized by the fact that it additionally comprises a diaphragm (11) that has an opening (21) that is arranged so that the light at the detection location is allowed to pass through the opening ( 21) while the light at a location other than the detection location is blocked by the diaphragm (11).
[0015]
15. Device according to claim 14, characterized by the fact that the diaphragm (11) additionally comprises at least one additional opening (18) which is arranged adjacent to the opening (21) when viewed in the direction of light propagation through the flat waveguide (2).
[0016]
16. System for detecting binding affinities characterized by the fact that it comprises a device, as defined in any of the preceding claims, and which additionally comprises a light source for emitting coherent light (1) of a wavelength predetermined, the light source and the device being arranged in relation to each other so that the coherent light (1) is coupled inside the flat waveguide (2) through the optical coupler (4).
[0017]
17. System according to claim 16, characterized by the fact that it additionally comprises an optical imaging unit (19), the optical imaging unit (19) being focused in order to produce an image of the detection location the device.
[0018]
18. The system according to claim 16, wherein the system is characterized by the fact that it additionally comprises a photodetector (20) for measuring the light intensity at the detection location.

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引用文献:

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

US4647544A|1984-06-25|1987-03-03|Nicoli David F|Immunoassay using optical interference detection|

JP3162448B2|1991-12-19|2001-04-25|松下電工株式会社|System ceiling|

DE59712897D1|1996-03-30|2008-01-03|Novartis Ag|INTEGRATED OPTICAL LUMINESCENCE SENSOR|

US6586193B2|1996-04-25|2003-07-01|Genicon Sciences Corporation|Analyte assay using particulate labels|

US6395558B1|1996-08-29|2002-05-28|Zeptosens Ag|Optical chemical/biochemical sensor|

SE514512C2|1999-07-02|2001-03-05|Proximion Fiber Optics Ab|Method and apparatus for coupling light|

KR100883079B1|1999-07-05|2009-02-10|노파르티스 아게|Sensor platform, apparatus incorporating the platform, and process using the platform|

US6510263B1|2000-01-27|2003-01-21|Unaxis Balzers Aktiengesellschaft|Waveguide plate and process for its production and microtitre plate|

US6951715B2|2000-10-30|2005-10-04|Sru Biosystems, Inc.|Optical detection of label-free biomolecular interactions using microreplicated plastic sensor elements|

US7264973B2|2000-10-30|2007-09-04|Sru Biosystems, Inc.|Label-free methods for performing assays using a colorimetric resonant optical biosensor|

EP1411816A4|2001-07-09|2005-09-28|Univ Arizona State|Affinity biosensor for monitoring of biological process|

JP2003194697A|2001-12-27|2003-07-09|Canon Inc|Explorer having grating coupler and its manufacturing method, probe having explorer, information processing device having probe, surface observation device, exposure device, and optical element by exposure device|

US6829073B1|2003-10-20|2004-12-07|Corning Incorporated|Optical reading system and method for spectral multiplexing of resonant waveguide gratings|

US7319525B2|2003-11-06|2008-01-15|Fortebio, Inc.|Fiber-optic assay apparatus based on phase-shift interferometry|

WO2005052644A2|2003-11-21|2005-06-09|Perkinelmer Las, Inc.|Optical device integrated with well|

EP1907805B1|2005-07-18|2012-10-10|Koninklijke Philips Electronics N.V.|Luminescence sensor using multi-layer substrate structure|

JP2008014732A|2006-07-04|2008-01-24|Tohoku Univ|Surface plasmon resonance measuring instrument|

US20100053610A1|2008-08-29|2010-03-04|Kwangyeol Lee|System and method for detecting molecules|

EP2741074A1|2012-12-04|2014-06-11|F. Hoffmann-La Roche AG|Device for use in the detection of binding affinities|

EP2757374A1|2013-01-17|2014-07-23|F. Hoffmann-La Roche AG|Method for preparing an outer surface of a planar waveguide to be capable of binding target samples along a plurality of predetermined lines and a planar waveguide|

EP2824446A1|2013-07-12|2015-01-14|F. Hoffmann-La Roche AG|Device for use in the detection of binding affinities|

EP2827130A1|2013-07-15|2015-01-21|F. Hoffmann-La Roche AG|Device for use in the detection of binding affinities|EP2824446A1|2013-07-12|2015-01-14|F. Hoffmann-La Roche AG|Device for use in the detection of binding affinities|

EP2827130A1|2013-07-15|2015-01-21|F. Hoffmann-La Roche AG|Device for use in the detection of binding affinities|

EP3040750A1|2014-12-29|2016-07-06|IMEC vzw|Device and method for performing lens-free imaging|

EP3862747A1|2015-07-07|2021-08-11|Furuno Electric Co., Ltd.|Measuring chip, measuring device, and measuring method|

KR101754774B1|2015-12-29|2017-07-06|주식회사 스칼라팍스트롯|Biochip and Method of manufacturing the Biochip|

EP3205512B1|2016-02-09|2018-06-13|CSEM Centre Suisse d'Electronique et de Microtechnique SA - Recherche et Développement|Optical security device|

WO2018143474A1|2017-02-06|2018-08-09|Tdk株式会社|Optical-waveguide-type sensor, and substance detection method|

DE102017211910A1|2017-07-12|2019-01-17|Dr. Johannes Heidenhain Gmbh|Diffractive biosensor|

US20200241301A1|2017-10-02|2020-07-30|Csem Centre Suisse D'electronique Et De Microtechnique Sa - Recherche Et Developpement|Resonant waveguide grating and applications thereof|

CN111788473A|2018-03-01|2020-10-16|弗·哈夫曼-拉罗切有限公司|Device for detecting binding affinity|

DE102019205527A1|2018-07-18|2020-01-23|Dr. Johannes Heidenhain Gmbh|Diffractive biosensor|

KR102080688B1|2018-10-23|2020-02-24|인하대학교 산학협력단|Grating coupler|

RU2700584C1|2018-12-13|2019-09-18|Общество С Ограниченной Ответственностью "Апто-Фарм"|Method for assessing the affinity of an oligonucleotide|

WO2021053042A1|2019-09-17|2021-03-25|F. Hoffmann-La Roche Ag|Biomolecular detection device|

DE102019134172A1|2019-12-12|2021-06-17|Dr. Johannes Heidenhain Gmbh|Device and method for coupling light with different wavelengths into a waveguide|

法律状态:
2018-12-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

2019-12-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

2020-06-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

2020-11-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/01/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

申请号 | 申请日 | 专利标题

EP12151436.8|2012-01-17|

EP12151436.8A|EP2618130A1|2012-01-17|2012-01-17|Device for use in the detection of binding affinities|

PCT/EP2013/050825|WO2013107811A1|2012-01-17|2013-01-17|Device for use in the detection of binding affinities|

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